Cyclic GMP Phosphodiesterase in Signaling

描述（由申请人提供）： 环核苷酸的细胞内水平的调节是细胞信号传导中的主要范例，特别是通过G蛋白偶联受体（GPCR）的信号传导。最近，我们已经表明，包括Frizzleds在内的7跨膜分段受体家族5的两个成员是与几个下游信号通路相关的GPCR。两个观察结果为该提议中的具体目标提供了基础：抑制小鼠F9畸胎瘤干细胞中的异源三聚体G蛋白亚基G（α）t2/o，以及用环GMP PDE抑制剂（如IBMX、扎匹司特和双嘧达莫）治疗，阻断GPCR大鼠Fz 2在钙瞬变和环GMP水平上发出信号的能力。我们已经创建了一种嵌合受体，其由众所周知的GPCR（β）2-肾上腺素能受体的跨膜配体结合结构域和外表面片段组成，三个胞内环（Ioops 1-3）和C-末端，Rfz 2的胞质尾已经被剪接到该外表面片段。这种新型嵌合体结合β-肾上腺素能激动剂和拮抗剂，向钙动员和环GMP细胞内浓度降低发出信号，但不像野生型（β）2-肾上腺素能受体那样向G（α）和腺苷酸环化酶发出信号。我们已经验证了该构建体的功能能力，并提出了两个具体的目的，以生物化学方式阐明G（α）t2/o和环GMP PDE在信号传导中的新作用：通过杆状病毒诱导的这些组分的表达和通过F9细胞中的BRET，测试Fz 2和在Sf 9细胞中表达的异源三聚体G蛋白之间的直接相互作用;并且，通过受体/G蛋白/PDE三联体的体外表达、纯化和重建，以及在F9细胞中使用反义抑制/拯救以及蛋白质-蛋白质相互作用的BRET分析的补充研究，来确定负责该信号传导的PDE的分子身份。我们将在这些细胞中采用一种新的药物诱导分泌系统来验证（与天然配体和受体）通过使用嵌合体所利用的观察。这些研究与信号传导以及阐明人类疾病的基础高度相关，其中信号传导途径的改变转化为异常生物学。

DESCRIPTION (provided by applicant): Regulation of intracellular levels of cyclic nucleotides is a major paradigm in cell signaling and, in particular, signaling by G-protein-coupled receptors (GPCRs). Recently, we have shown that two members of the family 5 of 7-transmembrane segmented receptors that includes Frizzleds are GPCRs with respect to several downstream signaling pathways. Two observations provide the basis for the specific aims in this proposal: suppression of the heterotrimeric G-protein subunits G(alpha)t2/o in mouse F9 teratocarcinoma stem cells and treatment with inhibitors of cyclic GMP PDE, such as IBMX, zaprinast, and dipyridamole, block the ability of the GPCR rat Fz2 to signal at the level of calcium transients and cyclic GMP. We have created a chimeric receptor composed of the transmembrane, ligand-binding domain and the exofacial segments of the well-known GPCR (beta)2-adrenergic receptor to which the three intracellular loops (iloops 1-3) and the C-terminal, cytoplasmic tail of Rfz2 have been spliced. This novel chimera binds beta-adrenergic agonists and antagonists, signaling to calcium mobilization and reduction in intracellular concentrations of cyclic GMP, but not to G(alpha)s and adenylylcyclase, like the wild-type (beta)2-adrenergic receptor. We have validated the functional capability of the construct and propose two specific aims to elucidate in a biochemical manner a new role for G(alpha)t2/o and cyclic GMP PDE in signaling: to test for the direct interaction between Fz2 and heterotrimeric G-proteins expressed in Sf9 cells biochemicaly by baculovirus-induced expression of these components and by BRET in F9 cells; and, to establish the molecular identity of the PDE(s) responsible for this signaling by expression, purification, and reconstitution of the triad of receptor/G-protein/PDE in vitro and complementary studies in F9 cells using antisense suppression/rescue as well as BRET analysis of protein-protein interactions. We shall employ a novel drug-induced secretion system in these cells to validate (with native ligand and receptor) the observations exploited by use of the chimera. These studies are highly relevant to signaling as well as to elucidation of basis for human diseases in which alterations in signaling pathways translate into aberrant biology.